department of pharmacy, faculty of science,...

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Identification of stereoisomeric metabolites of meisoindigo in rat liver

microsomes by achiral and chiral LC-MS/MS

Meng Huang, Lin Tang Goh, and Paul C. Ho

Department of Pharmacy, Faculty of Science, National University of Singapore,

Singapore (M. H., P.C.H.); Market Development & Applications, Waters Asia

Headquarter, Waters Asia Limited, Singapore (L.T.G.)

DMD Fast Forward. Published on August 18, 2008 as doi:10.1124/dmd.108.021956

Copyright 2008 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on August 18, 2008 as DOI: 10.1124/dmd.108.021956

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Running title: In vitro metabolism of meisoindigo

Address correspondence to: Paul C. Ho, Department of Pharmacy, National

University of Singapore, 18 Science Drive 4, Singapore 117543 Tel: +(65)

65162651 Fax: +(65) 67791554 Email: [email protected]

Number of text pages: 29

Number of tables: 4

Number of figures: 9

Number of references: 15

Number of words in the Abstract: 178

Number of words in the Introduction: 662

Number of words in the Discussion: 1208

Abbreviations: CML, chronic myelogenous leukemia; HPLC, high

performance liquid chromatography; UPLC, ultra performance liquid

chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry;

ESI, electrospray ionization; QTRAP, hybrid triple quadrupole linear ion trap;

QTOF, hybrid quadrupole time of flight; NMR, nuclear magnetic resonance; IR,

infrared.


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ABSTRACT:

N-methylisoindigotin, abbreviated as meisoindigo, has been a routine

therapeutic agent in the clinical treatment of chronic myelogenous leukemia in

China since the 1980s. However, information relevant to in vitro metabolism of

meisoindigo is limited. In this study, in vitro stereoisomeric metabolites of

meisoindigo in rat liver microsomes were identified for the first time, by achiral

and chiral liquid chromatography / tandem mass spectrometry, together with

proton NMR spectroscopy and synchrotron infrared spectroscopy. The major

in vitro phase I metabolites of meisoindigo were tentatively identified as

stereoselective reduced-meisoindigo, which comprised a pair of (3-R, 3’-R)

and (3-S, 3’-S) enantiomers with lower abundance, as well as another pair of

(3-R, 3’-S) and (3-S, 3’-R) enantiomers with higher abundance. One type of

minor in vitro metabolites were tentatively identified as stereoselective

N-demethyl-reduced-meisoindigo including a pair of (3-R, 3’-R) and (3-S, 3’-S)

enantiomers, as well as one meso compound. Another type of minor in vitro

metabolites were tentatively identified as both stereoselective and

regioselective monohydroxyl-reduced-meisoindigo. Based on the metabolite

profiling, three parallel metabolic pathways of meisoindigo in rat liver

microsomes were proposed.


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Introduction

Meisoindigo(3-(1,2-Dihydro-2-oxo-3H-indol-3-ylidene)-1,3-dihydro-1-methyl-2

H-indol-2-one), a synthetic derivative of indirubin from a traditional Chinese

medicine recipe, has been a routine therapeutic agent in the clinical treatment

of chronic myelogenous leukemia (CML) in China since 1980s (Cooperative

Group of Clinical Therapy of Meisoindigo, 1988; Xiao et al., 2000; Xiao et al.,

2002). A recent study indicated that the anti-angiogenesis effect of

meisoindigo may contribute to the anti-leukemic effect of this drug (Xiao et al.,

2006). Meisoindigo was equally efficient for both treated and previously treated

CML patients. The hematological complete response (CR) and partial

response (PR) rates were 45.0% and 39.3% for newly diagnosed patients and

35.9% and 41.4% for pretreated patients (Cooperative Group of Phase III

Clinical Trial on Meisoindigo, 1997). In addition, based on retrospective

analysis on the efficiency of different treatments for 274 CML patients followed

over a period of 5 years, meisoindigo in combination with hydroxyurea

significantly prolonged median duration of chronic phase and median survival,

as well as reduced incidence of blast crisis compared with meisoindigo or

hydroxyurea alone, which strongly indicated that meisoindigo has a synergistic

effect with hydroxyurea (Liu et al., 2000; Xiao et al., 2000). Meisoindigo was

generally well tolerated. The most frequent side effects were bone, joint and /

or muscle pain of varying degrees when the dosage was more than the

suitable dose. Approximately 30% of patients had mild nausea and vomiting.


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(Cooperative Group of Clinical Therapy of Meisoindigo, 1988; Cooperative

Group of Phase III Clinical Trial on Meisoindigo, 1997; Liu et al., 2000; Xiao et

al., 2000).

In order to improve the understanding of its efficacy and safety characteristics,

investigation of meisoindigo metabolism in animals or humans plays a critical

role. However, few publications relevant to this topic have been reported so far.

According to the literature survey information, only one individual study was

highly relevant to meisoindigo metabolism (Peng and Wang, 1990). In that

study, in vitro metabolism of meisoindigo in male rat liver microsomes was

investigated by reversed-phase high performance liquid chromatography

(RP-HPLC) with diode array detector (DAD). Based on the UV chromatogram

comparison of control and sample with parallel preparation, it was proposed

that ten chromatographic peaks were potential in vitro metabolites of

meisoindigo. Among them, two major chromatographic peaks were purified by

preparative thin layer chromatography (TLC) and predicted to be monohydroxy

metabolites based on preliminary electron impact (EI) MS results. However,

there are a few severe drawbacks with regard to its experimental design. The

substrate concentration adopted in that study far exceeded the upper limit of

the typical concentration range (10-50 μM) in metabolite identification

experiments (Chen et al., 2007). In addition, liquid-liquid extraction (LLE)

performed in that study was likely to cause the metabolites loss (Clarke et al.

2001), due to the fact that physicochemical properties of metabolites present


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are unknown in a certain extraction solvent or a mixed extraction solvent

system. Therefore, in vitro metabolism of meisoindigo in male rat liver

microsomes still need to be investigated with an appropriate experimental

design.

Diverse bioanalytical technologies have been applied in the field of drug

metabolism, such as radioimmunoassay (RIA), gas chromatography/mass

spectrometry (GC/MS), liquid chromatography (LC) with UV, fluorescence,

radioactivity or tandem mass spectrometry (MS/MS). Among these

technologies, liquid chromatography coupled with tandem mass spectrometry

(LC-MS/MS) has now become the most powerful tool owing to its superior

specificity, sensitivity and efficiency (Kostiainen et al., 2003). However,

LC-MS/MS alone does not always provide sufficient information for structural

characterization of metabolites, particularly in the aspects of identifying the

exact position of oxidation, differentiating isomers, or providing the precise

structure of unusual and/or unstable metabolites (Prakash et al., 2007). In

these cases, other analytical technologies such as nuclear magnetic

resonance (NMR), infrared (IR) or even chiral chromatography analysis have

to be utilized to supplement additional information.

In light of these considerations mentioned above, the purpose of the present

study was to qualitatively identify the in vitro metabolites of meisoindigo in rat

liver microsomes utilizing online LC-MS/MS and other relevant techniques if

necessary.


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Materials and Methods

Chemicals. All chemicals were of analytical grade and used without further

purification. Meisoindigo was provided by Institute of Materia Medica, Chinese

Academy of Medical Sciences and Peking Union Medical College, Beijing,

China. Bradford reagent, bovine serum albumin (BSA), β-NADPH, Dimethyl

sulfoxide (DMSO), ammonium acetate, palladium on carbon (Pd/C) and

Deuterochloroform (CDCl3) were purchased from Sigma Chemical Co. (St.

Louis, Mo, USA). HPLC grade methanol and acetonitrile were purchased from

Fisher Scientific Co. (Fair Lawn, NY, USA). Milli-Q water was obtained from a

Millipore water purification system (Billerica, MA, USA) and used to prepare

buffer solutions and other aqueous solutions.

Liver microsomal preparation. Pooled rat liver microsomes were prepared

according to the method reported previously (Gibson and Skett, 1994). Male

Sprague-Dawley (SD) rats were obtained from Animal Holding Unit, National

University of Singapore. Three rats were euthanized with CO2 after a 24-h

fasting period. The pooled extracted liver microsomes were suspended in

0.1M Tris buffer (PH 7.4) and stored at -80℃ before use. The microsomal

protein contents were determined by Bradford assay method, using BSA as

the standard protein (Bradford, 1976).

Liver microsomal incubations. Meisoindigo stock solution in DMSO was

added to 0.1M Tris buffer (pH 7.4) with rat liver microsomes. The mixture was

first shaken for 5min for equilibration in a shaking water bath at 37℃. The


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incubation was then initiated by adding β-NADPH solution. The final

concentrations of meisoindigo, NADPH and the microsomal protein were

50μM, 1mM and 1mg/mL, respectively in a typical incubation mixture (1mL).

The percentage of DMSO in the incubation mixture was kept less than 0.2 %

(v/v). Controls were prepared either by substituting microsomal protein

previously heated to 70-100℃ for 10 min or by replacing NAPDH with water.

Samples were incubated for 60 min and the reaction was terminated with the

same volume of ice-cold acetonitrile to precipitate proteins. Samples were

subsequently centrifuged at 14000 rpm for 15 min. The supernatant was taken

and analyzed by LC-MS/MS. All experiments were carried out in duplicate.

Achiral LC-MS/MS analysis.

HPLC-QTRAP. 5 μL of controls and samples were analyzed on a Q TRAP™

2000 LC-MS/MS system from Applied Biosystems / MDS Sciex (Concord,

Ontario, Canada) coupled to an Agilent 1100 HPLC system (Agilent

Technologies, Palo Alto, CA, USA). Separations were accomplished on a

Agilent hypersil ODS column (100 ×2.1 mm, 5 μm) (Agilent Technologies, Palo

Alto, CA, USA) with a guard cartridge at ambient temperature of about 22℃.

The mobile phase consisted of 10mM ammonium acetate in water (solvent A)

and methanol (solvent B) and was delivered at a flow rate of 0.2 mL/min. The

linear gradient elution program was as follows: 30% to 100% B over 12 min,

followed by an isocratic hold at 100% B for another 3 min. At 15 min, B was

returned to 30% in 1 min and the column was equilibrated for 19 min before the


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next injection. The total run time was 35 min. During LC-MS/MS analysis, up to

3 min of the initial flow was diverted away from the mass spectrometer before

evaluation of metabolites. The mass spectrometer was operated in the positive

ion mode with a TurboIonSpray source. The parameter conditions were

optimized as follows: curtain gas (CUR), 30 (arbitrary units); ion source gas 1

(GS1), 50 (arbitrary units); ion source gas 2 (GS2), 60 (arbitrary units); source

temperature (TEM), 300℃; declustering potential (DP), 26V; collision energy

(CE), 35eV; entrance potential (EP), 4.5V. Enhanced Q3 Single MS (EMS) and

Enhanced Product Ion (EPI) were utilized to obtain structural information of the

metabolites. The MS full scan range was set to m/z 50-350. The mass

spectrometer and the HPLC system were controlled by Analyst 1.4.1 software

from Applied Biosystems / MDS Sciex.

UPLC-QTOF. Accurate mass spectral data for the metabolites were obtained

with a Q-TOFTM Premier mass spectrometer (Waters Micromass, Manchester,

UK) coupled to an ACQUITY UPLCTM system (Waters Corporation, Milford,

MA, USA). The separations were performed on an ACQUITY UPLC BEH C18

column (100 × 2.1 mm, 1.7 μm). The column was maintained at 40 ℃ and

eluted with a mobile phase consisting of 0.1% formic acid in water (solvent A)

and 0.1% formic acid in methanol (solvent B) at 0.4 mL/min. The gradient

elution program was as follows: 30% to 95 % B over 6 min, followed by

increasing to 99 % B over 0.1 min. Then B was returned to 30 % in 0.1 min and

the column was equilibrated for 1.8 min before the next injection. The total run


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time was 8 min. The autosampler chamber was maintained at 10 ℃ and the

injection volume was 4 μL. The column eluent was directed to the mass

spectrometer for analysis. The mass spectrometer was equipped with an

electrospray ionization source and operated in the positive ionization mode.

Full-scan spectra were acquired from 50 to 1000 m/z with a scan time of 0.3 s.

The capillary and cone voltages were 3.0 kV and 25 V, respectively. The

source and desolvation temperatures were 90°C and 180°C, respectively. The

desolvation gas flow rate was 400 L/h and the cone gas flow rate was 10 L/h.

The collision energy was set at 4 eV. All analyses were acquired using an

independent reference spray via the LockSprayTM interference to ensure

accuracy and reproducibility. The [M+H]+ ion of sulfadimethoxine was

employed as the lock mass (m/z 311.0814). The LockSpray frequency was set

at 15 s. The accurate masses and compositions for the precursor ions were

calculated using the MassLynx V4.0 software incorporated in the instrument.

Search for metabolites. The total ion chromatograms (TIC) of controls and

samples for the EMS were first compared. The search for structure-related

metabolites of meisoindigo was performed manually by interpretation of mass

spectra and chromatograms. The possible molecular masses of metabolites

were screened out based on known mass shift of metabolites such as +16 for

hydroxylation. The corresponding EPI (MS/MS) of potential metabolites were

subsequently compared with that of meisoindigo to investigate the

fragmentation patterns and obtain information on the metabolite structures.


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Besides, MS/MS/MS of certain product ions gave more evidence about the

deduction of fragmentation mechanism. QTOF data also provided the accurate

masses and potential formulae to further confirm the structures of metabolites.

Isolation of metabolites by preparative HPLC and MS/MS/MS. The major

metabolites were isolated from a large-scale phenobarbital-induced rat liver

microsomal inbubation by Agilent 1100 preparative HPLC system (Agilent

Technologies, Palo Alto, CA, USA) on a Combi-A C18 column (50 × 20 mm, 5

μm) (Peeke Scientific, Redwood City, CA, USA) with a guard cartridge at

ambient temperature. The mobile phase consisted of water (solvent A) and

methanol (solvent B) and was delivered at a flow rate of 4 mL/min. The linear

gradient elution program was as follows: 30% to 100% B over 20 min, followed

by an isocratic hold at 100% B for another 5 min. At 25 min, B was returned to

30% in 1 min and the column was equilibrated for 4 min before the next

manual injection. The UV absorbance was monitored at 254 nm. All isolated

metabolites fractions were lyophilized and redissolved in 50% methanol/water

before chiral separation or direct infusion for MS/MS/MS analysis.

Chiral LC-MS/MS analysis. The rat liver microsomal samples and isolated

metabolite fractions were analyzed on a Q TRAP™ 3200 LC-MS/MS system

from Applied Biosystems / MDS Sciex (Concord, Ontario, Canada) coupled to

an Agilent 1200 HPLC system (Agilent Technologies, Palo Alto, CA, USA). The

volume of 20 μl was injected onto a Chiralcel OD-R column (250 × 4.6mm, 10

μm) (Daicel Chemical Industries, LTD., Japan) with a guard cartridge at


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ambient temperature. The isocratic mobile phase consisted of 20% water

(solvent A) and 80% methanol (solvent B) and was delivered at a flow rate of

0.4 mL/min. The total run time was 35 min. The mass spectrometer was

operated in the positive ion mode with a TurboIonSpray source. The parameter

conditions were optimized as follows: curtain gas (CUR), 20 (arbitrary units);

ion source gas 1 (GS1), 30 (arbitrary units); ion source gas 2 (GS2), 40

(arbitrary units); source temperature (TEM), 450℃; declustering potential (DP),

35V; collision energy (CE), 30eV; entrance potential (EP), 10V. Enhanced Q3

Single MS (EMS) was utilized to scan the mass range of m/z 50-700. The

mass spectrometer and the HPLC system were controlled by Analyst 1.4.2

software from Applied Biosystems / MDS Sciex.

Metabolite synthesis and purification. The starting material meisoindigo (50

mg) was dissolved in methanol (100 mL). After adding 10% palladium on

carbon (10 mg), the mixture was placed on the hydrogenator apparatus and

kept shaking at 40 psi for 2 h at room temperature. LC-MS was used to

monitor the consumption of starting material. The reaction mixture was filtered

through filter paper and the filter pad was washed with distilled water. The

filtrate was evaporated under reduced pressure and the precipitate was

purified by the same preparative HPLC and column described above. The

isocratic mobile phase consisted of 50% water (solvent A) and 50% methanol

(solvent B) and was delivered at a flow rate of 4 mL/min within 20 min. The UV

absorbance was monitored at 254 nm. All isolated metabolites fractions were


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evaporated under reduced pressure and stored under -20 ℃ before NMR

measurement.

NMR spectroscopy. Proton NMR spectra of synthesized metabolites were

recorded in CDCl3 at room temperature on a Bruker DRX 500 MHz NMR

spectrometer (Bruker BioSpin GmbH, Germany) using tetramethylsilane as an

internal standard.

Synchrotron IR spectroscopy. IR Spectra were recorded through grazing

incidence angle in reflection mode on a Bruker IFS 66v/S Fourier Transform

Infrared spectrometer (FTIR) (Bruker Optics GmbH, Germany) with a

synchrotron light source. The IR spectral range was from approximately 650

cm-1 to 4000 cm-1 with a resolution of 4cm-1. A KBr beamsplitter and mid band

MCT infrared detector cooled to 77K were used. The samples were dissolved

in acetonitrile and subsequently coated on gold substrate after solvent

evaporation. Spectra of thin film of samples on gold are directly acquired in the

vacuum.

Comparative study on incubations under normoxic and hypoxic

conditions. In normoxic incubation, meisoindigo stock solution in methanol

was added to 0.1M Tris buffer (pH 7.4) with rat liver microsomes. The mixture

was first shaken for 5 min for equilibration in a shaking water bath at 37℃. The

incubation was then initiated by adding β-NADPH solution. The final

concentrations of meisoindigo, NADPH and the microsomal protein were

10μM, 1mM and 1mg/mL, respectively in a typical incubation mixture (1mL).


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The percentage of methanol in the incubation mixture was kept less than 1%

(v/v). Aliquots of 100 µL of the incubation sample mixtures were collected at 5,

15, 30 and 60 minutes and the reaction was terminated with the same volume

of ice-cold acetonitrile to precipitate proteins. Samples were subsequently

centrifuged at 14000 rpm for 15 min. 5 μL of the supernatant was taken and

analyzed by LC-MS/MS. In hypoxic incubation, meisoindigo and rat liver

microsomes were preincubated at 37℃ and bubbled with 100% nitrogen gas

(the tip of the nitrogen gas supply was submerged approximately 2 mm in the

solution) for 5 min. The incubation was then initiated by adding β-NADPH

solution. The rest of the procedures for sample collection and preparation were

the same as described above except the continuous nitrogen gas supply

throughout the study. Negative controls were prepared either by substituting

microsomal protein previously heated to 70-100℃ for 10 min or by replacing

NAPDH with water under both normoxic and hypoxic conditions.

The analysis was conducted on a Q TRAP™ 3200 MS/MS system from

Applied Biosystems / MDS Sciex (Concord, Ontario, Canada) coupled to an

Agilent 1200 HPLC system (Agilent Technologies, Palo Alto, CA, USA).

Separations were accomplished on a Luna C18 column (150 × 2.0 mm, 5 μm)

(Phenomenex, Torrance, CA, USA) with a guard cartridge at ambient

temperature. The mobile phase consisted of 0.1 % formic acid in water

(solvent A) and acetonitrile (solvent B) and was delivered at a flow rate of 0.2

mL/min. The linear gradient elution program was as follows: 30 % to 95 % B


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over 13 min, followed by an isocratic hold at 95 % B for another 7 min. At 20

min, B was returned to 30 % in 1 min and the column was equilibrated for 19

min before the next injection. The total run time for each injection was 40 min.

The mass spectrometer was operated in the positive ion mode with a

TurboIonSpray source. Two multiple reaction monitoring (MRM) transitions i.e.

277.1→234.2 and 279.1→147.1 were used to determine meisoindigo and its

metabolites at m/z 279, respectively. The declustering potential (DP) and

collision energy (CE) values were 66 V and 45 eV for meisoindigo, 61 V and 30

eV for the metabolites. The other ionization parameters were as follows:

curtain gas (CUR), 20 (arbitrary units); ion source gas 1 (GS1), 40 (arbitrary

units); ion source gas 2 (GS2), 50 (arbitrary units); source temperature (TEM),

650 �; entrance potential (EP), 10 V. The dwell time of each MRM transition

was 150 msec. The mass spectrometer and the HPLC system were controlled

by Analyst 1.4.2 software from Applied Biosystems / MDS Sciex. The relative

percentages of the components in incubations were estimated at different

incubation time points based on the integrated peak areas of MRM

chromatograms.


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Results

The proposed fragmentation scheme and product ions of protonated

meisoindigo (m/z 277) are shown in Fig. 1. It contains major product ions at

m/z 249, 234, 218, 205,190,178, 132 and 104. Loss of CO (28 Da) following

gain of one H generated the product ion at m/z 249. Loss of CONH (43 Da)

following gain of one H generated the product ion at m/z 234. Loss of CONCH3

(57 Da) following loss of one H generated the product ion at m/z 218. Loss of

both CONH and CO generated the product ion at m/z 205. Loss of both

CONCH3 and CO following loss of one H generated the product ion at m/z 190,

which was confirmed by MS/MS/MS (data not shown). Loss of both CONCH3

and CONH following gain of two H generated the product ion at m/z 178, which

was confirmed by MS/MS/MS (data not shown). Cleavage of the 3,3’ double

bond of meisoindigo following gain of one H, and subsequent loss of CO at

position 2’ following gain of one H, generated the product ions at m/z 132 and

104, respectively.

The reconstructed extracted ion chromatogram (XIC) from HPLC-QTRAP was

shown in Fig. 2. It was found that the parent drug and its metabolites were

detected as their protonated molecules. Meisoindigo was eluted at 14.99 min

and showed a molecular ion (MH+) at m/z 277. Several metabolites were

observed and eluted earlier than meisoindigo, between 7 and 13 min. Those

metabolite peaks formed corresponding to masses of m/z 279 (11.19 min,

12.37 min), m/z 265 (9.30 min, 11.87 min), m/z 295 (7.97 min, 8.86 min, 9.37


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min, 10.34 min, 11.59 min) (summarized in Table 1). From the relative peak

areas in Fig. 2, meisoindigo remained the largest component, while two peaks

corresponding to m/z 279 and one peak corresponding to m/z 295 were

regarded as the major metabolites. The chromatographic profiles of

meisoindigo and its metabolites from UPLC-QTOF showed a significant

similarity as that from HPLC-QTRAP (data not shown). Table 2 showed the

accurate masses and potential molecular formulae provided by UPLC-QTOF.

Protonated metabolites at m/z 279. Metabolites at m/z 279 had retention

times of 11.19 min and 12.37 min on the HPLC system, respectively, and

showed 2 Da increase of molecular weight compared with parent drug,

suggesting they could be direct reduction products or N-demethylation

followed by mono-oxidation products. The corresponding potential formulae of

metabolites at m/z 279 based on their accurate masses was C17H15N2O2

(Table 2), thus confirming the occurrence of direct reduction. Taking the

identical molecular formulae with different retention times into account, these

two metabolites could be isomers.

Both of the EPI (MS/MS) mass spectra on metabolites at m/z 279 of 11.19 min

and 12.37 min were identical, indicating these two metabolites should be

stereoisomers. The proposed fragmentation scheme and MS/MS spectrum of

protonated metabolites at m/z 279 are shown in Fig. 3. Loss of H2O (18 Da)

generated the product ion at m/z 261. Loss of one or two CO generated the

product ion at m/z 251 or m/z 223, which indicated that reduction had not


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occurred on either of carbonyl groups. Loss of CONH generated the product

ion at m/z 234. Cleavage of the 3,3’ bond generated the product ions at m/z

147, and m/z 133 which was 1 Da higher than the corresponding product ion of

meisoindigo at m/z 132, thus indicating the presence of 3,3’ single bond.

Subsequent loss of CO at position 2 after the cleavage of the 3,3’ bond

generated the product ions at m/z 118, which was confirmed by MS/MS/MS

(data not shown).

Since direct double bond reduction caused the generation of two chiral carbon

centers located in position 3 and 3’, four stereoisomers i.e. M1, M2, M3, M4 at

m/z 279 could exist with possible absolute configurations of (3-R, 3’-R), (3-S,

3’-S), (3-R, 3’-S), (3-S, 3’-R). Among them, M1 and M2, M3 and M4 were

assumed as enantiomers of each other respectively. In addition, M1 was

diastereomer of M3 or M4. M2 was diastereomer of M3 or M4. Considering

enantiomers generally show the same chromatographic properties with the

use of non-chiral column while diastereomers seldom do, as well as only two

peaks were displayed on the TIC or XIC, the peak with retention time of 11.19

min was postulated to be a combination of M1 and M2, whereas the other peak

with retention time of 12.37 min was postulated to be a combination of M3 and

M4.

The chromatographic behaviors of metabolites at m/z 279 on the chiral column

further confirmed this postulation. The two peaks with retention time of 11.19

min and 12.37 min on non-chiral column (Fig. 2) were split into four peaks on


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chiral column, with retention times of 18.61 min, 21.44 min, 25.24 min and

27.69 min, respectively (Fig. 4 A). Interestingly, after separation of the two

pairs of enantiomers through preparative HPLC, one pair of enantiomer

(M1+M2) corresponded the leftmost and rightmost peaks with the retention

times of 18.70 min and 27.68 min (Fig. 4 B), whereas the other pair (M3+M4)

corresponded the two peaks in the middle with the retention times of 21.33 min

and 24.96 min (Fig. 4 C), which suggesting the chiral structure difference

between M1 and M2 was larger than that between M3 and M4. Furthermore,

the isolated pair of enantiomer (M1+M2) showed two additional small peaks

happening to correspond to the other pair (M3+M4) (Fig. 4 B), while the

isolated (M3+M4) only showed two peaks (Fig. 4 C), which strongly indicated

that (M1+M2) was unstable, automatically converting to (M3+M4). Hence the

absolute configurations of (M1+M2) could be (3-R, 3’-R) and (3-S, 3’-S). Based

on each stereoisomer’s chromatographic peak areas shown in Fig. 4 A, the

reduction metabolism of meisoindigo was found to be stereoselective. One pair

of enantiomer (M3+M4) showed a higher abundance than the other pair

(M1+M2). Within both of these two pairs of enantiomers, the more polar

stereoisomer eluted first was always of higher abundance than the less polar

one (18.70 min versus 27.68 min, 21.33 min versus 24.96 min).

The hydrogenation of red meisoindigo generated colorless products in

methanol solution. Diluted products showed the same retention times and

fragmentation patterns on LC-MS/MS as the two metabolites at m/z 279 from


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microsomal sample. After isolation by preparative HPLC and solvent

evaporation, two white solids of metabolites were obtained. The proton NMR

signals of meisoindigo, M1+M2, and M3+M4 were assigned in Table 3,

respectively. The significant similarity in proton NMR signals of M1+M2 and

M3+M4 further confirmed the diastereomer relationship between them.

Compared with the chemical shift values of meisoindigo, the chemical shift

values of both M1+M2 and M3+M4 more or less shifted to relatively high field

region, due to the breakdown of long conjugation within the whole molecule

caused by the forming of 3,3’ single bond. The synchrotron infrared absorption

spectra of two isolated metabolites at m/z 279 were very similar and shown in

Fig. 8 (A). The broad band around 3200-3400 cm-1 was assigned to be NH

stretching vibration. The band around 2800-2900 cm-1 was assigned to be CH3

stretching vibration. The band around 1600 cm-1 was assigned to be C=O

stretching vibration. These characteristic absorption bands further confirmed

the presence of NH, CH3 and C=O.

Therefore, M1+M2 was tentatively identified as a pair of (3-R, 3’-R) and (3-S,

3’-S) reduced-meisoindigo enantiomers with two hydrogens located at the

same side of 3,3’-single bond, whereas M3+M4 as another pair of (3-R, 3’-S)

and (3-S, 3’-R) enantiomers with two hydrogens located at the opposite sides.


times of 9.30 min and 11.87 min on the HPLC system, respectively, and

showed 12 Da decrease of molecular weight compared with parent drug,


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suggesting they could be direct reduction followed by N-demethylation

products. The corresponding potential formulae of metabolites at m/z 265

based on their accurate masses was C16H13N2O2 (Table 2), thus confirming

this postulation. Taking the identical molecular formulae with different retention

times into account, these two metabolites could be isomers.

Both of the EPI (MS/MS) mass spectra on metabolites at m/z 265 of 9.30 min

and 11.87 min were identical, indicating these two metabolites should be

stereoisomers. The proposed fragmentation scheme and MS/MS spectrum of

protonated metabolites at m/z 265 are shown in Fig. 5. Loss of H2O generated

the product ion at m/z 247, and subsequent loss of CO generated m/z 219.

Loss of one or two CO generated the product ion at m/z 237 or m/z 209, which

indicated that reduction had not occurred on either of carbonyl groups.

Cleavage of the 3,3’ bond generated the product ion at m/z 133 with the

highest abundance, indicating the forming of a symmetric molecule after

N-demethylation.

Due to the generation of two chiral carbon centers located in position 3 and 3’,

four stereoisomers i.e. M5, M6, M7, M8 at m/z 265 could exist with possible

absolute configuration of (3-R, 3’-R), (3-S, 3’-S), (3-R, 3’-S), (3-S, 3’-R).

Interestingly, reduction followed by N-demethylation led to the generation of

the same segments on the both sides of 3-3’ single bond. M5 (3-R, 3’-R), M6

(3-S, 3’-S) were enantiomers of each other so that they were combined into

one peak on the TIC or XIC with retention times of 9.30 min. However, M7 (3-R,


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3’-S), M8 (3-S, 3’-R) were actually the identical compound, due to the

presence of an internal symmetrical center at the midpoint of 3-3’ single bond.

M7 (namely M8) was a meso compound with retention times of 11.87 min.

Furthermore, M7 (M8) exhibited a higher abundance than the pair of

enantiomer (M5+M6) (Fig. 2), suggesting metabolites of m/z 265 also showed

stereoselective metabolism in this case.

Therefore, M5+M6 was tentatively identified as a pair of (3-R, 3’-R) and (3-S,

3’-S) N-demethyl-reduced-meisoindigo enantiomers with two hydrogens

located at the same side of 3,3’-single bond, whereas M7 (M8) as one meso

compound with two hydrogens located at the opposite sides.


times of 7.97 min, 8.86 min, 9.37 min, 10.34 min and 11.59 min on the HPLC

system, respectively, and showed 18 Da increase of molecular weight

compared with parent drug, suggesting they could be direct reduction followed

by mono-oxidation products. The corresponding potential formulae of

metabolites at m/z 295 based on their accurate masses was C17H15N2O3

(Table 2), thus confirming this postulation. Taking the identical molecular

formulae with different retention times into account, these five metabolites

could be isomers.

There were two types of EPI (MS/MS) mass spectra on metabolites at m/z 295.

One type was the identical EPI of metabolites with retention times of 9.37 min

and 10.34 min, indicating these two metabolites should be stereoisomers. The


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proposed fragmentation scheme and MS/MS spectrum of this type are shown

in Fig. 6. Loss of H2O generated the product ion at m/z 277. Loss of CO

generated the product ion at m/z 267. Loss of CONH generated the product

ion at m/z 251. Cleavage of the 3,3’ bond generated the product ions at m/z

163 and m/z 133, of which only m/z 163 was 16 Da higher than the

corresponding product ion of reduced-meisoindigo at m/z 147, thus indicating

hydroxylation had occurred at one of the positions 4, 5, 6 and 7. Subsequent

loss of CO at position 2 after the cleavage of the 3,3’ bond generated the

product ions at m/z 134. The other type was the identical EPI of metabolites

with retention times of 7.97 min, 8.86 min and 11.59 min, indicating these three

metabolites should be stereoisomers. The proposed fragmentation scheme

and MS/MS spectrum of this type are shown in Fig. 7. Loss of both H2O and

CO generated the product ion at m/z 249. Cleavage of the 3,3’ bond generated

the product ions at m/z 147 and m/z 149, of which only m/z 149 was 16 Da

higher than the corresponding product ion of reduced-meisoindigo at m/z 133,

thus indicating hydroxylation had occurred at one of the positions 4’, 5’, 6’ and

7’.

Reduction followed by phenyl single oxidation led to two different types of

position isomers. Considering the generation of two chiral carbon centers

located in position 3 and 3’, the first type could involve M9 (3-R, 3’-R), M11

(3-S, 3’-S), M13 (3-R, 3’-S), M15 (3-S, 3’-R), with single hydroxyl group at

position 4, 5, 6 or 7. The second type could involve M10 (3-R, 3’-R), M12 (3-S,


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3’-S), M14 (3-R, 3’-S), M16 (3-S, 3’-R), with single hydroxyl group at position 4’,

5’, 6’ or 7’. For each stereoisomer, there were four maximal possibilities

regarding the specific positions of hydroxyl group on the phenyl ring. In

addition, based on chromatographic peak areas shown in Fig. 2, the first type

i.e. M9, M11, M13, M15 showed a higher abundance than the second type i.e.

M10, M12, M14, M16, which suggested that the reduction followed by

mono-oxidation metabolism of meisoindigo was regioselective.

The synchrotron IR absorption spectra of metabolites at m/z 295 isolated by

analytical HPLC were very similar. The IR spectrum of the most concentrated

one was shown in Fig. 8 (B). The intensity of broad band around 3000-3500

cm-1 relative to the band around 2800-2900 cm-1 was higher compared with Fig.

8 (A), indicating the presence of both OH and NH. The OH stretching vibration

band was also found to be broad and slightly lower than NH stretching band.

The band around 2800-2900 cm-1 was assigned to be CH3 stretching vibration.

The band around 1600 cm-1 was assigned to be C=O stretching vibration.

These characteristic absorption bands further confirmed the presence of NH,

OH, CH3 and C=O.

Therefore, M9, M11, M13, M15 were within the bounds of (3-R, 3’-R), (3-S,

3’-S), (3-R, 3’-S), (3-S, 3’-R) monohydroxyl-reduced-meisoindigo isomers with

single hydroxyl group located at position 4, 5, 6 or 7, whereas M10, M12, M14,

M16 falling within another type of isomers with single hydroxyl group located at

position 4’, 5’, 6’ or 7’.


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Incubations under normoxic and hypoxic conditions. The relative

percentages of meisoindigo and its metabolites at m/z 279 in incubations with

rat liver microsomes at various incubation time points are given in Table 4. The

results showed that the difference of relative percentages between normoxic

and hypoxic incubations was the most significant at 5 min. Approximately

95.81 % of meisoindigo, 1.92 % of M1+M2, and 2.27 % of M3+M4 were found

in the 5-min incubation of meisoindigo under normoxic conditions, whereas

66.80 % of meisoindigo, 13.12 % of M1+M2, and 20.08 % of M3+M4 were

observed in the 5-min incubation of meisoindigo under hypoxic conditions,

thus indicating the occurrence of rapid reductive metabolism of meisoindigo

(Table 4). The difference of relative percentages between normoxic and

hypoxic incubations attenuated with time, until similar relative percentages

were found in the both normoxic and hypoxic incubations at 60 min (data not

shown). As a whole, a quicker onset of the metabolism under hypoxic

conditions was obtained as compared to that under normoxic conditions,

suggesting oxygen exposure strongly inhibits the in vitro reductive metabolism

of meisoindigo in rat liver microsomes.


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Discussion

This is the first investigation of the in vitro stereoisomeric metabolites of

meisoindigo in rat liver microsomes, by achiral and chiral LC-MS/MS, as well

as other relevant techniques. In the present study, rat liver microsomes were

chosen because rats express two major cytochrome P450 isoforms that are

oligomerically related to human CYP3A4, CYP3A1 and CYP3A2 (Nelson et al.,

1996). Besides, rat liver microsomes are readily available though simple

preparation. Although the metabolism study in human liver microsomes is not

involved yet in this study, the relevant results obtained showed a qualitative

similarity in metabolite profiles of meisoindigo between rat liver microsomes

and human liver microsomes (data not shown). In addition, the metabolite

profile of meisoindigo in rat liver microsomes is more comprehensive than that

in human liver microsomes (data not shown). Thus the findings regarding

metabolite profile in rat liver microsomes are of considerable importance.

The abundance of the minor metabolites is generally very low, especially in

incubation with low substrate concentration, but is increased relative to other

metabolites at higher substrate concentrations. Considering the typical

concentration range (10-50 μM) in metabolite identification experiments (Chen

et al., 2007), the upper limit concentration, i.e. 50 μM, was adopted.

Furthermore, minimal sample cleanup was performed, because the nature,

number, and concentrations of metabolites present were unknown, and it was


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therefore impossible to determine if any would be lost during sample

preparation (Clarke et al. 2001).

On the basis of molecular masses, fragmentation patterns, as well as potential

formulae of the metabolites and meisoindigo, three metabolic pathways of

meisoindigo in male rat liver microsomes were proposed as direct 3,3’ double

bond reduction, reduction followed by N-demethylation, as well as reduction

followed by phenyl mono-oxidation (Fig. 9). Among them, direct 3,3’ double

bond reduction was one-step reaction and thus the predominant pathway. The

other two parallel metabolic pathways were two-step reactions and thus

secondary pathways. Interestingly, the first two metabolic pathways of

meisoindigo were found to be stereoselective, while the third one was both

stereoselective and regioselective. In the cases of either M3+M4 versus

M1+M2 or M7(M8) versus M5+M6, the pair of (3-R, 3’-S) and (3-S, 3’-R)

enantiomer with two hydrogens located at the opposite sides showed a much

higher abundance than the other pair of (3-R, 3’-R) and (3-S, 3’-S) enantiomer

with two hydrogens located at the same sides.

Proton NMR spectroscopy nuclear Overhauser effect (NOE) experiments were

conducted to explore the NOE signal difference caused by distinct spatial

distance between 3H and 3’H within the two pairs of synthetic enantiomers,

namely M1+M2 and M3+M4. However, the results showed that there was no

significant difference in the NOE spectra between synthetic M1+M2 and

M3+M4, when 3H and 3’H were irradiated, respectively (data not shown).


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Other methods such as optical rotary dispersion (ORD), circular dichroism (CD)

or X-ray crystallography could be conducted to confirm the assignment of the

absolute configurations of M1+M2 and M3+M4. However, further investigation

on the specific absolute configurations of four stereoisomers i.e. M1, M2, M3,

M4 could be very challenging due to the rapid enantiomer interconversion

between M1 and M2, M3 and M4.

In terms of chiral chromatography analysis, a good separation of metabolite

stereoisomers could not be achieved through gradient elution mobile phase,

which is typically used for achiral chromatography analysis. Isocratic mobile

phase was finally adopted to successfully separate the two pairs of

enantiomers at m/z 279 simultaneously on a Daicel Chiralcel OD-R column.

But this chiral column didn’t work well on the separation of stereoisomeric

metabolites at m/z 265 and m/z 295, probably owing to their relatively low

quantity. Another possible reason might be the specific packing material of

Chiralcel OD-R column was only suitable for the chiral separation of

stereoisomeric metabolites at m/z 279 rather than the other two. In this case,

other different types of chiral columns could be considered.

Although the chromatographic peak at m/z 295 with retention time of 9.37 min

was regarded as the major metabolite, another peak of the pair of enantiomers

(M5+M6) with retention time of 9.30 min was almost co-eluted with it (Fig. 2).

More sensitive scan type multiple reactions monitoring (MRM) couple with

UPLC showed that this peak at 9.37 min in Fig. 2 was actually more than one


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component and combined with three individual peaks showing m/z 295 (data

not shown). Due to the chromatographic complexity and trace amount of these

minor metabolites, microsomal incubation samples had been directly tried on

the online LC-NMR 600 MHz equipped with a cryoprobe from Bruker BioSpin.

Unfortunately, injection volume was limited by the bulk concentration of

microsomal matrix contaminants in the first 5 minutes, which thus diluted the

eluted components entering into the NMR detection region. Analytical HPLC

was subsequently tried to isolate and accumulate the minor metabolites at m/z

295 and m/z 265 for further characterization by 1.7 mm Micro-CryoProbe NMR

600 MHz from Bruker BioSpin. However, the accumulated quantity was still

unable to meet the minimal requirement of this special NMR measurement

with the lowest detection of limit at present. Therefore, chemical synthesis has

to be the last resort. Further work would be needed to synthesize the possible

minor metabolites and isolate the stereoisomers by chiral chromatography

followed by NMR structural characterization. In particular, the specific position

of OH group in stereoisomeric monohydroxyl-reduced-meisoindigo is a vital

aspect to be investigated. Since both NCH3 at position 8 and NH at position 8’

donate electrons into the two phenyl ring systems, the highest electron density

should be located at ortho positions (i.e. 7 or 7’) and para positions (i.e. 5 or 5’).

In view of the offset by steric hindrance of OH group at ortho positions, it could

be predicted that para positions (i.e. 5 or 5’) would be thus the most reactive

towards perferryl oxygen complex [FeO]3+ from the perspective of CYP450


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oxidation mechanism.

Considering synchrotron IR spectroscopy provided a much greater detection of

limit than conventional IR technique due to the high brightness of synchrotron

IR source, it was employed for the first time to further confirm the structures of

metabolite within mg level after isolation and accumulation by analytical HPLC.

The IR measurement was performed in the vacuum to remove the signals

caused by H2O and CO2 from the air as much as possible, thus achieving a

satisfactory signal to noise ratio. It should be noted that synchrotron IR is only

applicable for those limited instances, in which the structural difference

between metabolite and parent drug is due to characteristic IR functional

groups, such as hydroxyl group, carbonyl group, amino group etc. Hence,

synchrotron IR can only be a supplementary tool to conventional structural

identification approaches, i.e., MS and NMR.

The comparative metabolite profiling using rat liver microsomes in the

normoxic and hypoxic incubations further confirmed the occurrence of

reductive metabolism of meisoindigo. Based on a report on the reductive

metabolism of meisoindigo analogues (Kohno et al., 2005), it is possible to

speculate that NADPH-cytochrome P450 reductase (P450R) in rat liver

microsomes could be the enzyme responsible for the initiation of reductive

activation of meisoindigo through the formation of a superoxide radical by

transferring one electron from the electron donor i.e. NADPH. This proposed

mechanism can be confirmed by investigating the metabolism of meisoindigo


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after incubation with purified recombinant P450R in the presence of NADPH.


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Acknowledgements. We thank Dr Mohammed Bahou, from Singapore

Synchrotron Light Source, National University of Singapore, for his kind

assistance with synchrotron IR experiments.


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References

Bradford MM (1976) A rapid and sensitive method for the quantitation of

microgram quantities of protein utilizing the principle of protein-dye binding.

Anal Biochem 72: 248-254.

Chen Y, Monshouwer M and Fitch LW (2007) Analytical tools and approaches

for metabolite identification in early drug discovery. Pharm Res 24: 248-257.

Clarke NJ, Rindgen D, Korfmacher WA and Cox KA (2001) Systematic LC/MS

metabolite identification in drug discovery. Anal Chem 73: 430A-439A.

Cooperative Group of Clinical Therapy of Meisoindigo (1988) Clinical studies

of meisoindigo in the treatment of 134 patients with CML. Chin J Hematol 9:

135-137.

Cooperative Group of Phase III Clinical Trial on Meisoindigo (1997) Phase III

clinical trial on meisoindigo in the treatment of chronic myelogenous leukemia.

Chin J Hematol 18: 69-72.

Gibson GG and Skett P (1994) Techniques and experiments illustrating drug

metabolism, in Introduction to Drug Metabolism (Gibson GG and Skett P eds)

pp 240-242, Blackie Academic and Professional, London.

Kostiainen R, Kotiaho T, Kuuranne T and Auriola S (2003) Liquid

chromatography/atmospheric pressure ionization-mass spectrometry in drug

metabolism studies. J Mass Spectrom 38: 357-372.

Liu BC, Qian LS, Wang Y, Xiao ZJ, Han JL, Li X, Zhou Z and Liu YH (2000)

The clinical effect and mechanism of meisoindigo in chronic granulocytic


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leukemia. Chin J Prac Intern Med 20: 610-612.

Kohno Y, Kitamura S, Yamada T, Sugihara K and Ohta S (2005) Production of

superoxide radical in reductive metabolism of a synthetic food-coloring agent,

indigocarmine, and related compounds. Life Sci 77: 601-614.

Nelson DR, Koymans L, Kamataki T, Stegeman JJ, Feyereisen R, Waxman DJ,

Waterman MR, Gotoh O, Coon MJ, Estabrook RW, et al. (1996) P450

superfamily: update on new sequences, gene mapping, accession numbers

and nomenclature. Pharmacogenetics 6: 1-42.

Peng Y and Wang MZ (1990) Screening for in vitro metabolites of meisoindigo

using RP-HPLC-DAD with gradient elution. Acta Pharmaceutica Sinica 25:

208-214.

Prakash C, Shaffer CL and Nedderman A (2007) Analytical strategies for

identifying drug metabolites. Mass Spectrom Rev 26: 340-369.

Xiao ZJ, Qian LS, Liu BC and Hao YS (2000) Meisoindigo for the treatment of

chronic myelogenous leukemia. Br J Haematol 111: 711-712.

Xiao ZJ, Hao YS, Liu BC and Qian LS (2002) Indirubin and meisoindigo in the

treatment of chronic myelogenous leukemia in China. Leuk Lymphoma 43:

1763-1768.

Xiao ZJ, Wang Y, Lu L, Li ZJ, Peng Z, Han ZC and Hao YS (2006)

Anti-angiogenesis effects of meisoindigo on chronic myelogenous leukemia in

vitro. Leuk Res 30: 54-59.


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Legends for Figures

FIG. 1. MS/MS spectrum of protonated meisoindigo at m/z 277 and the

proposed origin of key product ions.

FIG. 2. Reconstructed extract ion chromatogram for meisoindigo and its

metabolites in rat liver microsomes.

FIG. 3. MS/MS spectrum of protonated M1+M2, M3+M4 at m/z 279 and the


FIG. 4. Extracted ion chromatograms on the chiral column corresponding to

M1+M2+M3+M4 (A), M1+M2 (B) and M3+M4 (C) at m/z 279.

FIG. 5. MS/MS spectrum of protonated M5+M6, M7(M8) at m/z 265 and the


FIG. 6. MS/MS spectrum of protonated M9,M11,M13,M15 at m/z 295 and the


FIG. 7. MS/MS spectrum of protonated M10,M12,M14,M16 at m/z 295 and the



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FIG. 8. Infrared absorption spectra of mesoindigo metabolites at m/z 279 (A)

and m/z 295 (B).

FIG. 9. Proposed metabolic pathway of meisoindigo in rat liver microsomes.


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Tables

TABLE 1 HPLC-QTAP retention times and mass spectra properties for

meisoindigo and its in vitro metabolites

Retention time

(min) MH+ m/z of major product ions

meisoindigo 14.99 277 249,234,218,205,190,178,165,132,104

M1+M2 11.19 279 261,251,234,223,147,133,118

M3+M4 12.37

M5+M6 9.30 265 247,237,219,209,133

M7(M8) 11.87

M9,M11,M13,M15 9.37, 10.34 295

277,267,251,163,134,133

M10,M12,M14,M16 7.97, 8.86, 11.59 277,267,249,158,149,147


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TABLE 2 UPLC-QTOF retention times, potential formula and accurate masses for

meisoindigo and its in vitro metabolites

Retention time (min)

Molecular

Formula

(1+)

m/z (1+) Del M (Da) Measured m/z (1+)

PPM

meisoindigo 3.42 C17H13N2O2 277.0977 -0.0001 277.0976 -0.4

M1+M2 2.07 C17H15N2O2 279.1134

2.0162 279.1139 1.8

M3+M4 2.43 2.0153 279.1130 -1.4

M5+M6 1.65 C16H13N2O2 265.0977

-12.0005 265.0972 -1.9

M7(M8) 2.26 -11.9996 265.0981 1.5

M9,M11,M13,M15 1.66

C17H15N2O3 295.1083

18.0104 295.1081 -0.7

1.74 18.0111 295.1088 1.7

M10,M12,M14,M16

1.45 18.0111 295.1088 1.7

1.56 18.0113 295.1090 2.4

2.17 18.0101 295.1078 -1.7


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TABLE 3 1H-NMR data for meisoindigo, M1+M2, and M3+M4

Position Meisoindigo M1+M2 M3+M4

4 9.13 (1H, d, 8.0Hz) 7.26 (1H, t, 8.0Hz) 7.23 (1H, t, 8.0Hz)

5 7.08 (1H, m) 6.98 (1H, m) 6.95 (1H, m)

6 7.38 (1H, t, 7.5Hz) 7.15 (1H, m) 7.12 (1H, m)

7 6.79 (1H, m) 6.74 (1H, m) 6.72 (1H, m)

4’ 9.20 (1H, d, 8.0Hz) 7.33 (1H, t, 8.0Hz) 7.30 (1H, t, 8.0Hz)

5’ 7.05 (1H, m) 6.95 (1H, m) 6.92 (1H, m)

6’ 7.32 (1H, t, 7.5Hz) 7.08 (1H, t, 8.0Hz) 7.05 (1H, t, 8.0Hz)

7’ 6.81 (1H, m) 6.81 (1H, t, 8.5Hz) 6.78 (1H, m)

NCH3 3.30 (3H, s) 3.17 (3H, s) 3.14 (3H, s)

NH 7.81 (1H, br.s) 7.58 (1H, br.s) 7.48 (1H, br.s)

3 ⎯ 4.23 (1H, dd, 3.5Hz,

24.0Hz)

4.20 (1H, dd, 3.5Hz,

24.0Hz)

3’ ⎯ 4.32 (1H, dd, 3.5Hz,

26.0Hz)

4.30 (1H, dd, 3.5Hz,

27.0Hz)


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TABLE 4 Relative percentages of identified components in incubations of

meisoindigo with rat liver microsomes under normoxic and hypoxic conditions

Time

(min)

Normoxic (%) Hypoxic (%)

Meisoindigo M1+M2 M3+M4 Meisoindigo M1+M2 M3+M4

5 95.81 1.92 2.27 66.80 13.12 20.08

15 60.24 15.54 24.23 42.18 21.99 35.83

30 48.30 22.54 29.16 33.48 26.64 39.88


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department of pharmacy, faculty of science,...

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